Project Summary Single cell sequencing has revolutionized the way in which we define cell types and understand tissues, and has tremendous potential for analyzing the heterogeneity of complex tumors or large perturbation screens. At present, however, this powerful technology has serious limitations, particularly in what cellular information can be detected and analyzed. Current high-throughput microfluidics single cell RNA sequencing (scRNA-seq) methods can conveniently process tens of thousands of cells but only capture the extreme 3? ends of the most abundant transcripts from each cell. The low sensitivity and partial transcript coverage hampers these methods? abilities to detect allele-specific or subtle perturbation effects, while also dramatically increasing the per-cell sequencing cost. Moreover, no existing single-cell sequencing method can read DNA genotype and RNA expression from the same cell, which is crucial to studying non-coding regulatory elements and somatic rearrangements -- where the most genetic variation associated with cancer and human disease resides. Here we propose to overcome these limitations by developing a flexible high throughput single-cell sequencing system that (1) Can target many different regulatory DNA elements and ?transcripts simultaneously in the same cell?, (2) Is sensitive enough to measure subtle, allele-specific effects, (3) measures the effects of different mutations across tens-of-thousands of cells in a single assay, and (4) is easily and rapidly adaptable for application to any biological system with a heterogeneous cell population. We iteratively develop this technology, ensuring that each step independently creates new capabilities that address current scRNA-Seq limitations and enables allele specific expression analysis and perturbation screens of non-coding elements. First?, we will modify the the inDrop bead manufacturing process to make it flexible and rapidly customizable so that one large batch of universal barcoded beads can be conveniently adapted to target many specific transcript pools, SNP-containing portions of transcripts, and even genomic DNA, in just 8 hours. Second?, we test the sensitivity and allele-specificity of our new method in a predictably heterogeneous system: random X inactivation in hybrid female (XX) cells. Using single-molecule RNA-FISH as a gold standard, we will measure the sensitivity, specificity, and efficiency of our targeted scRNA-Seq approach. Third?, we enable simultaneous DNA genotyping and transcript quantification by adapting our custom beads and reaction conditions for isothermal amplification of genomic DNA loci, simultaneous with RT of targeted transcripts in the same cells. Fourth?, we will combine our approaches above in a proof-of-principle application to characterize enhancer function using a CRISPR mutagenesis screen. CRISPR mutagenesis randomly creates different alleles in each cell. We then use targeted sequencing of neighboring genes and DNA genotyping to evaluate the effect of each allele on its target(s) in ?cis?.